In the course of studying drug resistance in cancer, we and others identified a protein called t-Darpp (tDp) as a key component of the resistance phenotype. The normal function of tDp is completely unknown. Darpp-32 (Dp32) is a longer isoform of tDp that is well studied for its role in protein kinase A (PKA) signaling in dopaminergi neurotransmission. Dp32 works by protein-protein interaction; it is a dual-function protein that inhibits either protein phosphatase-1 (PP1) or PKA, depending on Dp32's phosphorylation status at residues T34 or T75. Its role in non-neuronal cells is unclear, but there is evidence to suggest that it might inhibit cell growth and migration. Our published studies suggest that tDp, the shorter protein, promotes cell proliferation (not inhibition) via PKA activation (not inhibitio). The molecular mechanism of this effect has not been determined, but it presumably also functions via protein-protein interaction. tDp lacks the N-terminal domain that is required for interaction with PP1, but phosphorylation at T75 (using the Dp32 numbering scheme) appears to be required for its activity in promoting cell proliferation. Thus, Dp32 and tDp seem to have antagonistic effects on cell growth and on PKA signaling, possibly via dominant-negative effects on each other's macromolecular interactions. We hypothesize that the physical and functional interaction of tDp, Dp32, and the PKA signaling network helps determine the balance between cell growth and growth inhibition. To address this hypothesis we need a greater understanding of Dp32 and tDp physical interactions in cells and the relationship between those interactions and protein function. Dissecting these interactions will require the contribution of multiple investigators with specialized expertise in molecular biology, cell biology, and protein biochemistry; the application of contemporary proteomic strategies and biophysical methods; and the development of innovative imaging-based approaches. We propose three specific aims: 1) Identify functionally- relevant macromolecular interactions for Dp32 and tDp in cell lysates; 2) Determine if functionally relevant macromolecular interactions occur in live cells; and 3) Determine the physical properties and molecular interfaces mediating Dp32 and tDp interactions.